The present invention relates to a method for substituting trityl, pixyl, and derivatives thereof for the hydrogen of a primary hydroxyl group. More particularly, the invention relates to a method for substituting trityl, pixyl, or derivatives thereof for the hydrogen of a support bound primary hydroxyl group, such as a hydroxyl group of a supported oligonucleotide.
In the chemical synthesis of polynucleotide fragments by the triester procedure, chromatographic methods are important for the purification of protected intermediates and for the isolation of the unprotected final product. The chromatographic behavior of the oligomers can be varied within certain limits by protecting groups, generally producing an unspecific change in the total polarity of the molecule. For example, Gortz et al. Angew. Chem. Int. Ed. Engl., 20(8): 681-683 (1981) describe introducing 4-alkoxytrityl groups for hydrophobic protection at the 5'-end of oligonucleotides. These 4-alkoxytrityl groups contain 8, 10, 12, 14, and 16 carbon atoms. Gortz et al. synthesized 4-hexadecyloxytrityl protected thymidine by first treating 4-hexadecyloxytritanol with acetyl chloride to produce 4-hexadecyloxytrityl chloride. The 4-hexadecyloxytrityl chloride was then treated with thymidine in anhydrous pyridine in the presence of 4-dimethylaminopyridine. After various purification and separation procedures, a 75% yield was obtained. This 4-hexadecyloxytrityl protected thymidine was then condensed onto the 5'--O--position of the terminal nucleotide of a support bound oligonucleotide.
A disadvantage of the procedure of Gortz et al. is that in order to practice this procedure for attaching a hydrophobic protecting group to support bound oligonucleotide, it is necessary to maintain a supply of four separate protected nucleosides, namely, 4-alkoxytrityl protected thymidine, 4-alkoxytrityl protected adenosine, 4-alkoxy protected cytosine, and 4-alkoxytrityl protected guanosine. This is because support bound oligonucleotides can have as a terminal nucleotide any one of these four nucleosides. Accordingly, this requires the outlay of funds for four separate reagents.
A further disadvantage of the Gortz et al. technique is that a 75% yield can be inadequate for many applications.
Moreover, as detailed in the example section below, it has been discovered that the Gortz et al. procedure is ineffective for support bound nucleotides.
Therefore, it would be very desirable if one could obtain a higher yield upon adding the desired group to the nucleoside. It would also be very desirable if a single reagent could be employed when such a group is inserted on the support bound oligonucleotide.